This application is the national phase under 35 U.S.C. xc2xa7371 of PCT International Application No. PCT/FI99/00204 which has an International filing date of Mar. 17, 1999, which designated the United States of America.
A The present invention relates to novel matrix metalloproteinase (MMP) inhibitors and down-regulators, to a process for the preparation of these inhibitors, to pharmaceutical compositions comprising these inhibitors/downregulators, to the use of the novel matrix metalloproteinase inhibitors for the manufacture of pharmaceutical and research preparations, to a method for inhibiting and down-regulating MMP-dependent conditions either in vivo or in vitro, to a method for inhibiting formation, synthesis, expression and/or functions as well as actions of matrix metalloproteinases, and to the use of the novel MMP inhibitors in biochemical isolation and purification procedures of matrix metalloproteinases.
Matrix metalloproteinases (MMPs) constitute a superfamily of genetically closely related proteolytic enzymes capable of degrading almost all the constituents of extracellular matrix and basement membrane that restrict cell movement. MMPs also process serpins, cytokines and growth factors as well as certain cell surface components (Woessner, 1991; Birkendal-Hansen, 1995; Chandler et al., 1997). MMPs are thought to have a key role in mediating tissue remodeling and cell migration during morphogenesis and physiological situations such as wound healing, trophoblast implantation and endometrial menstrual breakdown. MMPs are further involved in processing and modification of molecular phenomena such as tissue remodeling, angiogenesis, cytokine, growth factor, inte grin and their receptor processing (Chandler et al., 1997). MMPs also mediate release and membrane-bound proteolytic processing of tumor necrosis factor (TNF-xcex1) by bacterial-virulence factor induced monocytes. This event is mediated by a membrane-bound metalloproteinase TACE (TNF-xcex1 activating enzyme). Thus MMP-inhibitors, such as the novel peptides presented in this invention, can i.a. prevent activation of TNF-xcex1 by blocking this type of activating enzymes (Shapira et al., 1997).
Several studies have shown that the expression and activities of MMPs are pathologically elevated over the body""s endogenous anti-proteinase shield in a variety of diseases such as cancer, metastatis, rheumatoid arthritis, multiple sclerosis, periodontitis, osteoporosis, osteosarcoma, osteomyelitis, bronchiectasis, chronic pulmonary obstructive disease, and skin and eye diseases. Proteolytic enzymes, especially MMPs, are believed to contribute to the tissue destruction damage associated with these diseases.
There is a variety of other disorders in which extracellular protein degradation/destruction plays a prominent role. Examples of such diseases include arthritides, acquired immune deficiency syndrome (AIDS), burns, wounds such as bed sores and varicose ulcers, fractures, trauma, gastric ulceration, skin diseases such as acne and psoriasis, lichenoid lesions, epidermolysis bollosa, aftae (reactive oral ulcer), dental diseases such as periodontal diseases, peri-implantitis, jaw and other cysts and root canal treatment or endodontic treatment, related diseases, external and intrinsic root resorption, caries etc.
At least 20 members of the MMP-superfamily are known (Birkendal-Hansen, 1995; Pei and Weiss, 1996; Llano et al., 1997), and the number of MMP-family members and their cellular origins is growing all the time. Each of the MMP enzymes contains a putative tridentate Zn2+ binding site which is believed to constitute the active site in the enzyme. Very recently, three new members of the MMP-family were discovered by screening cDNA libraries for homologies to conserved regions of the known MMP genes and named the membrane-type matrix metalloproteinases-1, -2, and -3 (MT-MMP-1, -2, and-3). Based on their predicted amino acid sequences, each of the MT-MMPs like almost all previously characterized MMPs, contains (i) a candidate leader sequence, (ii) a propeptide region which includes a highly conserved PRCGXPD(SEQ ID NO:12) sequence that helps to stabilize the MMP zymogen in a catalytically inactive state, (iii) a zinc-binding catalytic domain, and (iv) a hemopexin-like domain near their respective C-termini. In addition, in a pattern similar to that described for stromelysin-3, each of the MT-MMPs contains a short amino acid insert sandwiched between their pro- and catalytic domains that encodes a potential recognition motif for members of the proprotein convertase family. Despite their considerable similarity to other MMP family members, however, only the MT-MMPs contain approximately 75-100 amino acid extensions at their C-termini, each of which includes a hydrophobic stretch consistent with the presence of a transmembrane (TM) domain. Thus, in contradistinction to all other MMPs, the MT-MMPs are expressed as membrane-associated ectoenzymes rather than soluble proteins (Pei and Weiss, 1996).
A comprehensive review of the MMP-family members, their activation, modes of action, their inhibition by various natural proteins (endogenous inhibitors) and synthetic compounds as well as details of the involvement of MMP family members in various pathological conditions and diseases is given by Woessner (1991); Krane (1994); Birkendal-Hansen et al. (1993); and Birkendal-Hansen (1995), the whole disclosures of which are incorporated herein by reference. In the scope of the present invention the term matrix metalloproteinase (MMP) refers to all discovered MMPs.
The gelatinase A or 72 kDa MMP-2 and gelatinase B or 92 kDa MMP-9 were originally described as type IV collagenases because they appeared to be essential enzymes for the degradation of the basement membrane (Tryggvason et al., 1987). Cells need to traverse the endothelial basement membrane during entry to and exit from the circulation. This is also a critical key step in the metastatic cascade tumor cells have to accomplish before they can metastasize to distant organs. MMP-2 and MMP-9 may also have a function in other steps of the metastatic cascade such as in angiogenesis (Hanahan and Folkman, 1996; Volpert et al., 1996) and local tumor invasion (Stetler-Stevenson et al., 1993).
Because MMPs are potential targets for therapeutic intervention, much work has been focused on the design of synthetic metalloproteinase inhibitors. Many MMP-inhibiting compounds containing reactive zinc-chelating groups such as thiol, hydroxamate, EDTA, phosphonamidate, phosphinate etc. have been developed (Beckett et al., 1996). Some of the peptidomimetics have shown beneficial effects in animal models of metastasis, arthritis, and other inflammatory diseases. Tumor cell invasion can also be inhibited by the native MMP inhibitors TIMP-1 (tissue inhibitor of metalloproteinase) and TIMP-2. MMPs can also be inhibited by peptides based on the highly conserved prodomain region of MMPs that is important for latency of MMPs (Park et al., 1991; Melchiori et al., 1992; Fotouhi et al., 1994). In addition, tetracyclines and their nonantimicrobial chemically-modified (CMT) as well as anthracycline derivatives have been found to inhibit MMPs (Golub et al., 1992; Sorsa et al., 1994).
Although the above discussion shows that some inhibitors for MMPs do exist and have been investigated, the tests are still mostly at the experimentation stage and no clinically acceptable inhibitor for MMPs exists as a therapeutic or prophylactic drug for any of the pathological states and diseases potentially connected with MMPs. Adverse side effects which have been detected in the above described MMP inhibitors include, for instance, toxicities (synthetic peptides), antimicrobial activities (tetracyclines), etc.
An alternative to rational molecular design is to screen libraries of random peptides or other chemicals to find lead compounds binding to target molecules. In particular, peptide libraries displayed on the surface of bacteriophage have often yielded valuable binding peptides to target proteins. However, it has been more difficult to isolate inhibitors to proteinases from libraries of short peptides, possibly because short peptides are easily degraded by proteinases. Phage-displayed peptide libraries have rather been utilized to obtain information of the sequences cleaved by a proteinase (Matthews and Wells, 1993; Smith et al., 1995). Inhibitors to proteinases have been developed with phage surface expression and selection of large proteinase inhibitor domains in which certain active site residues have been randomized (Roberts et al., 1992; Dennis et al., 1995).
The present inventors have now succesfully isolated novel peptide inhibitors to MMPs, especially to MMP-9 and MMP2, using phage-displayed libraries of peptides that were conformationally restrained by designed disulfide bonds. The most active MMP inhibitors developed are capable of inhibiting in vitro migration of endothelial cells as well as invasion of tumor cells, thus being potential lead compounds to design peptidomimetics to block MMPs. The peptides can also be used in column chromatographic matrices for biochemical isolation and purification procedures of MMPS.
It is therefore an object of the present invention to provide novel matrix metalloproteinase inhibitors and binding-ligands based on the cyclic structure (disulfide bond between cysteines) of the peptide motif
CXXHWGFXXC
which corresponds to the sequence shown in SEQ ID No. 1 of the sequence listing and wherein X is any amino acid residue.
It is another object of the present invention to provide novel matrix metalloproteinase inhibitors and down-regulators based on the cyclic structure of the peptide motifs
CRRHWGFEFC
which corresponds to the sequence shown in SEQ ID No. 2, and
CTTHWGFTLC
which corresponds to the sequence shown in SEQ ID No. 3.
The present invention also relates to a pharmaceutical composition comprising an amount of the novel matrix metalloproteinase inhibitor(s)/down-regulator(s) effective to reduce the activities, activations, functions, and/or expressions of one or more MMPs, especially of MMP-2 and/or MMP-9, and a pharmaceutically and biochemically acceptable carrier. Pharmaceutical compositions comprising novel MMP inhibitor(s)/down-regulator(s) according to the invention may be used systemically, locally and/or topically. They also include all potential combinations (combo-medications) with other MMP-inhibitors, other drugs and tumor-homing chemicals/molecules.
The present invention also includes the use of the novel matrix metalloproteinase inhibitors for the manufacture of pharmaceutical preparations for the treatment of matrix metalloproteinase dependent conditions, and also their use, for example as affinity ligands, in biochemical purification and isolation procedures of MMPs. The MMP-dependent conditions include, but are not limited to, wounds, burns, fractures, lesions, ulcers, cancer and metastasis progression in connective tissues and bone, periodontitis, gingivitis, peri-implantitis, cysts, root canal treatment, internal and external root canal resorption, caries, AIDS, corneal ulceration, gastric ulceration, aftae, trauma, acne, psoriasis, loosening of the endosseal hip-prosthesis, osteomyelitis, osteoporosis, tissue remodeling, angiogenesis, arthritides (rheumatoid, reactive and osteo arthritides), angiogenesis, lung diseases (bronchiectasis and chronic obstructive pulmonary diseases and other lung diseases).
The present invention also relates to a process for the preparation of novel matrix metalloproteinases which process comprises standard solid-phase Merrifield peptide synthesis.
The novel CXXHWGFXXC(SEQ ID NO:1) structure according to the invention does not show similarity to previously described MMP inhibitors, although the activities of CTTHWGFTLC(SEQ ID NO:3) resemble the properties of chemically modified tetracyclines (CMTs) as will be described below. The peptides comprising the novel structure were derived from the single cysteine-expressing CX9 library and exhibited a HWGF(SEQ ID NO:13) consensus sequence. All contained a second cysteine showing a cyclic structure CXXHWGFXXC(SEQ ID NO:1). Phage attachment experiments indicated that the cloned phages bound to MMP-9 with considerable affinity.
The cyclic peptides according to the invention inhibited degradation of gelatin and casein substrates by MMP-2 and MMP-9 with IC50 of 5-10 xcexcg/ml. Of a series of peptides synthesized, the HWGF(SEQ ID NO:13)-containing peptides CRRHWGFEFC(SEQ ID NO:2) and CTTHWGFTLC(SEQ ID NO:3) were found to be most promising as inhibitors of MMP-9. These two HWGF(SEQ ID NO:13)-containing peptides also inhibited MMP-2. The fact that the peptides were selected on MMP-9 but can strongly inhibit also MMP-2 indicates that the peptides recognize a binding site very similar between MMP-9 and MMP-2.
The most active HWGF(SEQ ID NO:13)-containing peptide developed (CTTHWGFTLC)(SEQ ID NO:3) inhibited cell migration studied in normal serum-containing media, and blocked the migration of human endothelial cells as well as invasion of HT1080 fibrosarcoma and C8161 melanoma cells through a reconstituted basement membrane. These findings imply that both cancer cells and endothelial cells may use quite a similar MMP-dependent mechanism to migrate that is sensitive to the down-regulating effect of CTTHWGFTLC(SEQ ID NO:3). The high activity of CTTHWGFTLC(SEQ ID NO:3) could at least partially be due to the fact that the peptide can not only inhibit an active enzyme but can interfere with the autoactivation of purified proMMP-9 and proMMP-2 as is shown below by using gelatin and casein substrates. The peptide can also down-regulate the production of MMP-9. In contrast to the phage binding data in which we were unable to see any phage binding to proMMP-9, the synthetic CTTHWGFTLC(SEQ ID NO:3) peptide does bind to proMMP-9 as indicated by single-step isolation of proMMP-9 from human leukocyte buffy coats using affinity chromatography with the peptide coupled to Sepharose. On the whole, it is possible that by binding to proMMPs the peptide can hinder the true proteolytic activation by other proteinases that is the likely activation mechanism during cell invasion.
The corresponding linear peptides were virtually inactive as demonstrated by a loss of activity after reduction and alkylation of the cysteines. Especially preferred MMP inhibitors according to the present invention are thus the cyclic peptide inhibitors CTTFIWGFTLC(SEQ ID NO:3) and CRRHWGFEFC(SEQ ID NO:2), which inhibit the activity of MMP-2 and MMP-9 as shown below.
As stated above, the novel cyclic peptide inhibitors we have developed are useful lead compounds to design peptidomimetics to block MMPs and cell migration. The CXXHWGFXXC(SEQ ID NO:1) motif may also be utilized to develop more selective inhibitors to individual members of the MMP family, as MMP-2 and MMP-9 were differently inhibited by the two CXXHWGFXXC(SEQ ID NO:1) peptides: MMP-2 was more strongly inhibited by CTTHWGFTLC(SEQ ID NO:3) while MMP-9 was preferentially inhibited by CRRHWGFEFC(SEQ ID NO:2). Selective inhibitors directed e.g. to MMP-2 might be more efficient in preventing tumor dissemination, as in many experimental systems the metastatic potential of tumor cells rather correlated with MMP-2 activity rather than with MMP-9 activity. Finally the small size of the MMP-targeting cyclic peptides can be utilized to carry drugs to tumors. Phage-library derived peptides targeting receptors in tumor vasculature have been found to be useful cytotoxic drug carriers to tumors in mice. MMPs are potential receptors for targeted chernotherapy, because they are usually overexpressed in tumors as compared to normal tissues and appear to be involved in the angiogenic process.
Thus, as a result of the invention, MMP dependent conditions may now be treated or prevented either with the novel MMP inhibitors alone or in combination with other drugs normally used in connection with the disease or disorder in question. These include for example tetracyclines, chemically modified tetracyclines (Golub et al., 1992), bisphosphonates, as well as homing/carrier molecules to the sites of tumors, such as integrin-binding peptides (Arap et al., 1998). The amount of novel matrix metalloproteinase inhibitors to be used in the pharmaceutical compositions according to the present invention varies depending on the specific inhibitor used, the patient and disease to be treated as well as the route of administration.
The novel MMP inhibitors of the present invention have shown no toxicity when injected into animals and do not affect cell number or viability as determined by trypan blue dye exclusion.
The present invention thus also relates to a method for the therapeutic or prophylactic treatment of MMP-dependent conditions in mammals by administering to said mammal an effective amount of the novel MMP-inhibitor(s), as well as to a method for inhibiting the formations, synthesis, expressions, activations, functions and actions of MMPs in mammals by administering the novel MMP-inhibitor(s)/down-regulator(s) in an amount which is effective in blocking the formation, activation and actions of MMPs.
The present invention also relates to a method for inhibiting matrix metalloproteinases in vitro comprising adding to an in vitro system the novel matrix metalloproteinase inhibitor(s) in an amount which is effective in inhibiting the MMP activity.
A further object of the invention is a method for isolating and purifying matrix metalloproteinases with the aid of the novel matrix metalloproteinase inhibitor(s).
FIG. 1 shows the results from the inhibition of MMP-9-mediated [125I]-gelatin degradation using synthetic peptides. APMA-activated MMP-9 was preincubated with the CRRHWGFEFC(SEQ ID NO:2) and CTTFIWGFTLC(SEQ ID NO:3) at the concentrations indicated for 1 h before adding [125I]-gelating substrate. After gelatinolysis for 1h, the counts released into medium were determined. The results show means from duplicate measurements. Similar results were obtained in three independent experiments.
FIG. 2 shows gelatinolysis induced by APMA-activated MMPs or their proforms. The concentrations of the cyclic and linear CRRHWGFEFC(SEQ ID NO:2) peptide were 10 xcexcg/ml. The results show means from duplicate experiments.
FIG. 3 shows inhibition of MMP-2-mediated casein degradation by CTTHWGFTLC(SEQ ID NO:3) (A, B) and CRRHWGFEFC(SEQ ID NO:2) (C, D). After 1 h pretreatment with the peptides, APMA-activated MMP-2 (A, C) or proMMP-2 (B, D) was incubated with the casein for 2 h. 52 xcexcM xcex2-casein was used as substrate for MMPs. Shown is Coomassie Blue-staining of the 21 kD xcex2-casein (lane 1) and its fragments (lanes 2-9) resolved by SDS-PAGE (A, B); CTTHWGFTLC(SEQ ID NO:3) was used at the concentrations of (2) 0 xcexcg/ml, (3) 75, (4) 50, (5) 25, (6) 10, (7) 5, (8) 1, and (9) 0.5 xcexcg/ml in the lanes 2-9, respectively. (C, D); the concentrations of CRRHWGFEFC(SEQ ID. NO:2) were 0, 250, 100, 50, 25, 10, 1, and 0.5 xcexcg/ml, respectively.
FIG. 4 shows binding of proMMP-9 to CTTHWGFTLC(SEQ ID NO:3) peptide coupled to Sepharose. Lysate of human buffy coat cells was applied to each peptide Sepharose, and the bound proteins were analyzed on SDS gels followed by Coomassie Blue staining (lanes 1-2), or immunoblotting with anti-MMP-9 antibodies (lanes 5-6). Lanes 1 and 5 show proteins eluted from CTTHWGFTLC(SEQ ID NO:3)-Sepharose. Lanes 2 and 6 show proteins eluted from GACLRSGRGCGA(SEQ ID NO:5)-Sepharose. Lane 3 shows protein staining of the cell lysate. Lane 4 displays the molecular weight markers of 200, 92, 76, and 55 kDa.
FIG. 5 shows how CTTHWGFTLC(SEQ ID NO:3) inhibits migration of HT1080 fibrosarcoma cells. Cells were pretreated with CTTHWGFTLC(SEQ ID NO:3) at the concentrations indicated or with 500 xcexcg/ml of the unrelated EVGTGSCNLECVSTNPLSGTEQ(SEQ ID NO:4) control peptide for 2 h. Cells were plated on transwell chambers and allowed to migrate for 20 h in 10% serum-containing medium. Cells that traversed to the undersurface of the filter were stained and the filter area was scanned. The results show mean optical densityxc2x1S.D. from triplicate wells. The optical density of blank Transwell without cells was of 0.000.
FIG. 6 shows comparison of the efficacy of the MMP inhibitors CTTHWGFTLC(SEQ ID NO:3) and CMT-8 to prevent migration of C8161 melanoma cells. Cells were pretreated with CTTHWGFTLC(SEQ ID NO:3), CMT-8, or with the EVGTGSCNLECVSTNPLSGTEQ(SEQ ID NO:4) control, and allowed to migrate 20 h in Transwell chambers. Cells that migrated to the undersurface of the filter were stained and scanned. The results show mean optical densityxc2x1S.D. from triplicate wells.
FIG. 7 shows inhibition of endothelial cell migration by CTTHWGFTLC(SEQ ID NO:3). Endothelial cells were allowed to migrate for 18 h in the presence of 20% serum for HUVEC, or 10% serum for Eahy92 line. Shown is the relative number of cells having traversed to the undersurface of Transwell chambers. The results show meansxc2x1SD from triplicate wells.
FIG. 8 shows inhibition of Matrigel invasion of tumor cells by CTTHWGFTLC(SEQ ID NO:3). C8161 or HT1080 cells were allowed to invade through Matrigel for 24 h in 10% serum-containing medium. The concentrations of CTTHWGFTLC(SEQ ID NO:3) and the EVGTGSCNLECVSTNPLSGTEQ(SEQ ID NO:4) control were 500 xcexcg/ml. The invaded cells were counted, and the relative number of cells are expressed as meansxc2x1S.D. from triplicate wells.
FIG. 9 shows that breast carcinoma growth is clearly inhibited by CTTHWGFTLC(SEQ ID NO:3) peptide.
FIG. 10 shows gelatin zymography of melanoma cell conditioned medium. CTTHWGFTLC(SEQ ID NO:3) peptide but not the control peptide inhibits the formation of active 82 kD MMP-9.
FIGS. 11A, 11B and 11C show MB-435 breast carcinoma cells grown for 2 days in 10% serum in the absence or presence of CTTHWGFTLC(SEQ ID NO:3) peptide.
FIG. 12 shows the effect of CTTHWGFTLC(SEQ ID NO:3) (P291) on keratinocyte gelatinase production and expression.
FIG. 13 shows the effect of CTTHWGFTLC(SEQ ID NO:3) (P291) on keratinocyte migration. The photograph of the plates is taken after 4 days of migration.